Cardiovascular disease (CVD) has consistently been the leading cause of death in the United States for nearly a century. Adverse outcomes of CVD usually result from rupturing atherosclerotic plaques. The most effective strategy for combating CVD is secondary prevention: diagnosis and treatment of subclinical disease. Non-invasively assessing atherosclerotic inflammation, the cause of plaque instability, is an important element of this strategy. Traditional imaging modalities cannot assess plaque vulnerability and thus are of limited predictive value. In contrast, molecular imaging visualizes the anatomy together with the biochemical signature of plaques. Current molecular imaging techniques used for CVD have setbacks, e.g., high cost and inherent risk to the patient. Bioluminescence imaging (BLI) does share these problems: oxidation-sensitive probes such as luminol and its derivative L-012 emit harmless, visible-spectrum light upon contact with reactive oxygen species (ROS) produced by phagocytes. These phagocytes are enriched in the arterial walls of CVD patients, mainly in and around inflamed plaques, and directly contribute to their rupture. The goal of this proposal is to develop BLI to detect ROS produced by these cells in animal models, thus allowing atherosclerotic inflammation to be imaged in vivo. This goal has been focused into three Specific Aims: In Aim 1, the chemiluminescence properties of three known BLI probes will be analyzed for use in single- cell bioluminescence microscopy (BLM). First, each probe will be tested for ROS specificity and chemical signal enhancement potential. The reproducibility of these findings in cellular environments will be tested with phagocytes isolated from animal models. Optimized conditions determined by these experiments will be used to visualize individual phagocytes with BLM as they initiate the ROS-synthesis cascade. In Aim 2, BLI will be used to measure inflammation of animal models both ex vivo and in vivo. First, BLI will be evaluated as an inexpensive alternative to current clinical assays for serum myeloperoxidase, a soluble enzyme marker for CVD and that creates the most potent physiological ROS, hypochlorous acid. Next, luminol derivative L-012 will be used to image atherosclerosis in apolipoprotein E-deficient mice. BLI signal will be correlated with plaque inflammation as quantified by immunohistochemistry. Aim 3 consists of a plan to synthesize new BLI probes that have improved properties for use in vivo, namely red-shifted emission spectra that are capable of penetrating deeper tissues. The proposed conjugates, oxazine and squaraine derivates, will be evaluated against current compounds in experiments similar to those outlined in Aims 1 and 2. The scaffolds of these conjugates will allow for a large number of variants to be synthesized with the possibility of becoming next-generation BLI probes.